Treatment of AIDS with antibodies to gamma interferon, alpha interferon and TNF-alpha

ABSTRACT

The present disclosure concerns the treatment of a patient with autoimmune disease, including AIDS, by neutralizing, removing or inhibiting different types of interferons, tumor necrosis factor, HLA class II antigens, IgE, and other pathological factors and/or their receptors, as well as neutralizing, removing or inhibiting autoantibodies, including antibodies to target cells, CD4 cells and DNA. Treatment comprises administration of an autoimmune inhibitor, or extracorporeal exposure of the patient&#39;s fluid to an immunosorbent comprising an autoimmune inhibitor, followed by return of the treated fluid to the patient; or it comprises a combined therapy involving extracorporeal immunosorption in conjunction with the administration of an autoimmune inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is divisional of U.S. application Ser. No. 09/306,286filed on May 6, 1999, now abandoned, which is a continuation of U.S.application Ser. No. 08/995,730, filed on Dec. 22, 1997, now U.S. Pat.No. 6,333,032, which is a continuation of U.S. application Ser. No.08/771,831, filed on Dec. 23, 1996, now U.S. Pat. No. 5,888,511 which isCIP of Ser. No. 08/025,408 filed Feb. 26, 1993 now U.S. Pat. No.5,626,843.

FIELD OF INVENTION

The present invention is a method to treat autoimmune diseases andconditions in a patient, which are caused by the disturbance of thesynthesis of interferons (IFNs) and certain other substances (e.g. tumornecrosis factors (TNFs)) and the production of autoantibodies to targetcells, including CD4 cells, which damage the patient's immune system andhave a direct pathological action on the patient's cells.

BACKGROUND OF THE INVENTION

The ability of the immune system to discriminate between “self” and“non-self” antigens is vital to the functioning of the immune system asa specific defense against invading microorganisms. “Non-self” antigensare those antigens on substances entering or in the body which aredetectably different or foreign from the animal's own constituents,whereas “self” antigens are those which, in the healthy animal, are notdelectably different or foreign from its own constituents. However,under certain conditions, including in certain disease states, anindividual's immune system will identify its own constituents as“non-self,” and initiate an immune response against “self” material, attimes causing more damage or discomfort as from an invading microbe orforeign material, and often producing serious illness in an individual.Autoimmune disease results when an individual's immune system attackshis own organs or tissues, producing a clinical condition associatedwith the destruction of that tissue, as exemplified by diseases such asrheumatoid arthritis, insulin-dependent diabetes mellitus, acquiredimmunodeficiency syndrome (“AIDS”), hemolytic anemias, rheumatic fever,Crohn's disease, Guillain-Barre syndrome, psoriasis, thyroiditis,Graves' disease, myasthenia gravis, glomerulonephritis, autoimmunehepatitis, multiple sclerosis, systemic lupus erythematosus, etc.Blocking, neutralizing or inhibiting the immune response or removing itscause in these cases is, therefore, desirable.

Autoimmune disease may be the result of a genetic predisposition, aloneor as the result of the influence of certain exogenous agents such as,viruses, bacteria, or chemical agents, or from the action of both. Someforms of autoimmunity arise as the result of trauma to an area usuallynot exposed to lymphocytes, such as neural tissue or the lens of theeye. When the tissues in these areas become exposed to lymphocytes,their surface proteins can act as antigens and trigger the production ofantibodies and cellular immune responses which then begin to destroythose tissues. Other autoimmune diseases develop after exposure of theindividual to antigens which are antigenically similar to, that iscross-reactive with, the individual's own tissue. For example, inrheumatic fever an antigen of the streptococcal bacterium, which causesrheumatic fever, is cross-reactive with parts of the human heart. Theantibodies cannot differentiate between the bacterial antigens and theheart muscle antigens, consequently cells with either of those antigenscan be destroyed.

Other autoimmune diseases, for example, insulin-dependent diabetesmellitus (involving the destruction of the insulin producing beta-cellsof the islets of Langerhans), multiple sclerosis (involving thedestruction of the conducting fibers of the nervous system) andrheumatoid arthritis (involving the destruction of the joint liningtissue), are characterized as being the result of a mostly cell-mediatedautoimmune response and appear to be due primarily to the action ofT-cells (See, Sinha et al., Science 248: 1380 (1990)). Yet others, suchas myesthenia gravis and systemic lupus erythematosus, are characterizedas being the result of primarily a humoral autoimmune response (Id.).Nevertheless, the autoimmune diseases share a common underlyingpathogenesis, resulting in the need for safe and effective therapy. Yetnone of the presently available drugs are completely effective for thetreatment of autoimmune disease, and most are limited by severetoxicity.

In recent years, a new point of view on the pathogenesis of autoimmunediseases, including AIDS, has developed, in which it has been suggestedthat autoimmune disease is connected with a disturbance in the synthesisof interferons and other cytokines induced by interferons (Skurkovich etal., Nature 217:551-2 (1974); Skurkovich et al., Annuals of Allergy35:356 (1975); Skurkovich et al., J. IFN Res. 12, Suppl. 1:S110 (1992);Skurkovich et al. Med. Hypoth. 41:177-185 (1993); Skurkovich et al.,Med. Hypoth. 42:27-35 (1994); Gringeri et al., Cell. Mol. Biol41(3):381-387 (1995); Gringeri et al. J Acquir. Immun. Defic. Syndr.13:55-67 (1996)). IFN has been found in the circulation of patients withautoimmune diseases, and it has been neutralized in vivo with antibodyto leukocyte (alpha) IFN (“IFNα”). Healthy people do not have interferonin their blood (Skurkovich et al., 1975). In addition, it has been shownthat hyperproduced IFNα is found not only in the circulation of patientswith classic autoimmune diseases, but also in patients with HIVinfection (DeStefano et al., J. Infec. Disease 146:451 (1982)), whereits presence is a predictive marker of AIDS progression (Vadhan-Raj etal., Cancer Res. 46:417 (1986)). The IFN induced by HIV has lowanti-(HIV) viral activity (Gendelman et al., J. Immunol. 148:422(1992)). It was shown that the circulating IFNα possesses antigenicspecificity like natural IFNα, which is pH stable, but this interferonis pH labile like IFNγ (Preble et al., Science 216:429 (1982)); thus, itis known as aberrant IFNα.

Investigators have also shown that tumor necrosis factors (TNFα andTNFβ) also play a significant role in the pathology of autoimmunediseases. For example, the presence of TNFα has been correlated withrheumatoid arthritis (RA) (Brennan et al., Brit. J. Rheum. 31(5):293-8(1992)), and TNFα has been found to be related to an increase in theseverity of collagen induced arthritis in animal models (Brahn et al.,Lymphokine and Cytokine Res. 11(5):253 (1992)), while it has also beenshown that anti-TNF alpha antibody administration ameliorates collageninduced arthritis (Williams et al. Clin. & Exp. Immunol 87(2):183(1992)). TNFα is increased in the serum of PA patients (Holt et al.Brit. J. Rheum. 21(11):725 (1992); Altomonte et al., Clin. Rheum.11(2):202 (1992), and both the cytokine (Chu et. al., Brit. J. Rheum.31(10):653-661 (1992)) and its receptors have been identified inrheumatoid synovium, as well as at the cartilage-pannus junction(Deleuran et al., Arthritis Rheum. 35(10)1180 (1992)).

In addition, increased circulating levels of TNFα have been found to beassociated with disease progression in patients with multiple sclerosis(Shariff et at., N. Engl. J. Med. 325(7):467-472 (1992)); whileincreased serum levels of soluble TNF receptor and interferon γ (“INFγ”)have been independently correlated with disease activity in individuals,e.g., those with systemic lupus erythematosus (Aderka et. al., ArthritisRheum. 36(8): 1111-1120 (1993); Machold et al., J. Rheumat.17(6):831-832 (1990)). The spontaneous release of interferon and TNF inHIV-positive subjects (Vilcek et al., In AIDS: The Epidemic of Karposi'sSyndrome and Opportunistic Infections, A. E. Friedman-Kien & L. J.Laubenstein, eds. Masson Publishing, New York, N.Y., 1986; Hess et al.,Infection 19, Suppl 2:S93-97 (1991); Biglino et al., Infection 19(1):11/7-11/7 (1991)), and the decline seen in the serum levels of TNF-αin RA patients following long term administration of the diseasemodifying drug sulfasalazine (Danis et al., Ann. Rheum. Disease51(8):946 (1992)), further suggest that the concentrations of cytokinesand/or their receptors is reflected in the clinical course of autoimmunedisease.

IFN is known to induce tumor necrosis factor (TNF) and its receptors(Lau et al. AIDS Research and Human Retroviruses 7:545 (1991)), whichenhances virus replication (Matsuyama et al., Proc. Natl. Acad. Sci. USA86:2365 (1989)). In addition to its presence in the circulation, IFNshave also been found in the cerebrospinal fluid in some patients withpsychiatric mid neurologic diseases (Lebikoa et al., Acta. Biot MedGerm. 38:879 (1979); Preble et al, Am. J. Psychiatry, 142:10 (1985)), aswell as in patients with rheumatoid arthritis. Therefore, since healthypeople do not have interferons in their spinal or synovial fluids, theinventors have suggested that one or more alpha IFNs may be involved inthe development of the initial autoimmune disease response.Consequently, the removal and/or neutralization of IFNα has beenproposed as a method of treatment of patients with autoimmune disease,including AIDS. The appearance of cytokines and autoimmunogens inducedby IFNα and their prolonged circulation in the body is an inseparablepart of the development of autoimmune disease, triggering immunedysregulation in autoimmune disease, including AIDS. See, U.S. Pat. Nos.4,824,432; 4,605,394; and 4,362,155, herein incorporated by reference.However, it now appears that gamma IFN (“IFNγ”) can also play apathogenetic role since each participates in immune regulation.

In addition to classic autoimmune disease and AIDS, autoantibodies playa pathogenic role in many other pathological conditions. For example,after cell (or organ) transplantation or after heart attack or stroke,certain antigens from the transplanted cells (organs) or necrotic cellsfrom the heart or the brain can stimulate the production ofautoantibodies or immune lymphocytes (Johnson et al., Sem. Nuc. Med.19:238 (1989); Leinonen el al., Microbiol. Path. 9:67 (1990); Montalbanet al., Stroke 22:750 (1991)), which later participate in rejection (inthe case of a transplant) or attack cardiac or brain target cells,aggravating the condition. Moreover, in human autoimmune disease certaincells express abnormally elevated levels of HLA class II antigens, whichis stimulated by the disturbed production of cytokines, e.g., IFNγalone, or IFNγ in combination with TNF (Feldman et al. “Interferons andAutoimmunity,” In IFN 9, Academic Press, p. 75 (1987).

Recognition of the important role of cytokines in autoimmune disease hasfostered the development of a new generation of therapeutic agents tomodulate cytokine activity. Preliminary results of trials in whichanti-interferon polyclonal antibodies were administered to a small groupof rheumatoid patients suggest improvement in both the clinical and thelaboratory manifestations of the disease (Skurkovich et al., Annals ofAllergy 39:344-350 (1977)). Moreover, proteins, such as polyclonalantibodies and soluble receptors targeted against interferons and TNF-αare currently being evaluated in clinical trials for the treatment of RAand other autoimmune diseases. The administration of monoclonalantibodies to TNF-α has provided encouraging early results in thetreatment of patients with severe RA (Elliott et. al., J. Cell.Biochem., Suppl 17B: 145 (1993); Elliott et al., Lancet 344: 1105-1110(1994)). Also positive preliminary results were achieved in AIDSpatients given antibodies or other agents to reduce the level ofcirculating IFNα in the body (Skurkovich et al., 1994; Gringeri et al.1996). However, because autoimmune diseases are complex, oftencharacterized by multiple cytokine abnormalities, effective treatmentappears to require the simultaneous administration or utilization ofseveral agents, each targeting a specific cytokine pathway or itsby-product. To meet this need, the methods of treatment of the presentinvention include not only the use of specific antibodies, but also,provide pleiotrophic autoimmune inhibitors, including antibodies tocytokines and HLA class II antigens, and antigens for the removal ofautoantibodies to target cells or DNA. The use of these antibodies andantigens as disclosed in the present invention results in the removal,neutralization or inhibition of the pathogenic cytokine(s), HLA class IIantigens, and/or autoantibody(ies) to target cells or DNA from theautoimmune patient, thereby significantly improving the quality of lifeof the individual.

SUMMARY OF THE INVENTION

The present invention concerns the treatment of autoimmune diseases,including AIDS, by blocking, inhibiting, neutralizing, or removingharmful interferons, tumor necrosis factors, and other pathologicalimmunogens or factors, or their receptors, and antibodies to targetcells, including CD4 cells, in a patient in need of such treatment.

It is an object of the present invention to provide a method of treatingautoimmune disease in a patient comprising administering to the patientan effective amount of anti-IFNγ antibodies and/or antibodies to IFNγreceptor.

In addition, it is an object of the invention to provide a method oftreating autoimmune disease in a patient comprising administering to thepatient an effective amount of a plurality of at least two antibodiesselected from the group consisting of anti-IFNα antibodies andantibodies to IFNα receptor, anti-IFNγ antibodies and antibodies to IFNγreceptor, anti-TNF antibodies and antibodies to TNF receptor, andantibodies to an HLA class II antigen or its receptor. In particular, itis an object of the invention to provide a method of treating autoimmunedisease, wherein a plurality of antibodies is administered comprising(a) at least one anti-IFNα antibody and/or antibody to IFNα receptor,and (b) at least one anti-IFNγ antibody and/or antibody to IFNγreceptor. It is also a particular object of the invention to provide amethod of treating autoimmune disease, wherein at least one anti-TNFantibody and/or antibody to TNF receptor is administered, alone or inconjunction with one or more of the following: anti-IFNα antibody,antibody to IFNα receptor, anti-IFNγ antibody and/or antibody to IFNγreceptor. It is further a particular object of the invention to providea method of treating autoimmune disease, wherein at least one antibodyto an HLA class II antigen and/or its receptor is administered, alone orin conjunction with one or more of the following: anti-IFNα antibody,antibody to IFNα receptor, anti-IFNγ antibody, antibody to IFNγreceptor, anti-TNF antibody and/or antibody to TNF receptor.

It is also an object of the invention to provide a method of treatingautoimmune disease in a patient comprising, in conjunction withadministering to the patient an effective amount of a plurality of atleast two antibodies selected from the group consisting of anti-IFNγantibodies and antibodies to IFNα receptor, anti-IFNγ antibodies andantibodies to IFNγ receptor, anti-TNF antibodies and antibodies to TNFreceptor, and antibodies to an HLA class II antigen or its receptor, anextracorporeal treatment comprising removing antigens from the patientby drawing fluid from the patient; passing said fluid through animmunosorbent comprising a combination of at least two antibodiesselected from the same group; followed by returning the treated fluid tothe patient. In particular, it is an object of the invention to providea method of treating autoimmune disease, wherein the extracorporealtreatment comprises passing the patient's fluid through an immunosorbentcomprising a combination of at least two antibodies selected from (a) atleast one anti-IFNα antibody and/or antibody to IFNα receptor, and (b)at least one anti-IFNγ antibody and/or antibody to IFNγ receptor. It isalso a particular object of the invention to provide an extracorporealmethod of treatment, wherein the immunosorbent comprises at least oneanti-TNF antibody and/or antibody to TNF receptor, alone or inconjunction with one or more of the following: anti-IFNα antibody,antibody to IFNα receptor, anti-IFNγ antibody and/or antibody to IFNγreceptor. It is further a particular object of the invention to providean extracorporeal treatment, wherein the immunosorbent comprises atleast one antibody to an HLA class II antigen and/or its receptor, aloneor in conjunction with one or more of the following: anti-TFNα antibody,antibody to IFNα receptor, anti-IFNγ antibody, antibody to IFNγreceptor, anti-TNF antibody and/or antibody to TNF receptor.

Yet another object of the invention is to treat specific autoimmunediseases by the administration of autoimmune inhibitor to the patient.For example, a method of treatment is provided comprising administeringto the patient an effective amount of beta interferon in addition to oneor more antibodies selected from the group consisting of anti-IFNαantibody and antibodies to IFNα receptor, anti-IFNγ antibodies andantibodies to IFNγ receptor, anti-TNF antibodies and antibodies to TNFreceptor, and antibodies to an HLA class II antigen or its receptor.This method is particularly effective for the treatment of multiplesclerosis. An additional method of treatment is provided comprisingadministering to the patient an effective amount of antibodies tointerleukin (“IL”), preferably to IL-6, in addition to one or moreantibodies selected from the group consisting of anti-IFNα antibodiesand antibodies to IFNα receptor, anti-IFNγ antibodies and antibodies toIFNγ receptor, anti-TNF antibodies and antibodies to TNF receptor, andantibodies to an HLA class II antigen or its receptor. This method isparticularly effective for the treatment of systemic lupus erythematosusand insulin-dependent diabetes mellitus.

It is also an object of the invention to provide a method of treatingspecific autoimmune diseases by the extracorporeal exposure of thepatient's fluid to an immunosorbent comprising autoimmune inhibitor,followed by the return of the treated fluid to the patient. This methodmay be practiced alone, or in conjunction with the administration ofautoimmune inhibitor to the patient. For example, a method of treatmentis provided comprising exposing the patient's fluid to an immunosorbentcomprising an effective amount of antibodies to interleukin, preferablyanti-IL-6 antibody, in addition to one or more antibodies selected fromthe group consisting of anti-IFNα antibody and antibodies to IFNαreceptor, anti-IFNγ antibodies and antibodies to IFNγ receptor, anti-TNFantibodies and antibodies to TNF receptor, and antibodies to an HLAclass II antigen or its receptor. This method is particularly effectivefor the treatment of systemic lupus erythematosus and insulin-dependentdiabetes mellitus.

It is a further particular object of the invention to provide a methodof treating specific autoimmune diseases, wherein the specificextracorporeal treatment comprises passing the patient's fluid throughan immunosorbent comprising antibody (ies) to immunoglobulin E (“IgE”),followed by the return of the treated fluid to the patient. This methodmay be practiced alone, or in conjunction with the administration ofautoimmune inhibitor. For example, for treating certain diseases relatedto hypersensitivity of the immediate type, e.g., bronchial asthma,antibody to IgE is used as an immunosorbent, alone or in conjunctionwith other autoimmune inhibitors, such as antibodies to IFNs and/or toTNF or to their receptors.

It is a further object of the invention to provide a method of treatingautoimmune disease in a patient comprising, alone or in conjunction withadministering to the patient an effective amount of one or moreantibodies (e.g., anti-IFNα antibodies, antibodies to IFNα receptor,anti-IFNγ antibodies, antibodies to IFNγ receptor, anti-TNF antibodies,antibodies to TNF receptor, and antibodies to an HLA class II antigen orits receptor), extracorporeal treatment comprising removingautoantibodies from the patient by drawing fluid from the patient;passing said fluid over anti-TNF antibodies comprising an effectiveamount of different target cells, CD4 cells and/or DNA, to remove,neutralize or inhibit auto-antibodies in the patient's fluid; followedby returning the treated fluid to the patient. The immunosorbent forextracorporeal treatment may further comprise one or more antibodies(e.g., anti-IFNα antibodies, antibodies to IFNα receptor, anti-IFNγantibodies, antibodies to IFNγ receptor, anti-TNF antibodies, antibodiesto TNF receptor, and antibodies to an HLA class II antigen or itsreceptor.

In particular, it is an object of the invention to provide a method oftreating specific autoimmune diseases, wherein the specificextracorporeal treatment comprises passing the patient's fluid throughan immunosorbent comprising target cells. For example, for the treatmentof rheumatoid arthritis, target cell antigens from joints, skin,collagen, and possibly other target antigens are used as immunosorbents,alone or in conjunction with other autoimmune inhibitors, such asantibodies to IFNs and/or antibodies to TNF or their receptors and/orantibodies to HLA class II antigens or their receptors. In addition, forthe treatment of rheumatic fever, the invention provides animmunosorbent comprising antibodies to IFNs and/or to TNF and/or theirreceptors and/or to HLA class II antigens and/or their receptors and/orother substances, in conjunction with a second cardiac tissue sorbentfor removing autoantibodies against cardiac tissue. The second sorbentcan also include selected serotypes of Streptococcus (group “A”),because certain antigens from cardiac tissue and some serotypes ofStreptococcus are antigenically similar. For the treatment of autoimmunediseases of the central nervous system, target cell antigens from thebrain cells are used to absorb autoantibodies formed against braincells.

It is also a particular object of the invention to provide a method oftreating specific autoimmune diseases, wherein the specificextracorporeal treatment comprises passing the patient's fluid throughan immunosorbent comprising DNA. For example, for the treatment ofsystemic lupus erythematosus the immunosorbent comprises DNA to remove,reduce or neutralize the patient's anti-DNA autoantibodies.

Yet another particular object of the invention to provide a method oftreating specific autoimmune diseases, wherein the specificextracorporeal treatment comprises passing the patient's fluid anti-TNFantibodies comprising CD4 cells. For example, for the treatment of AIDS,the immunosorbent comprises CD4 cells, alone or in conjunction withother autoimmune inhibitors, such as antibodies to IFNs and/or TNFand/or HLA class II antigen, or their receptors.

It is also an object of the invention to provide pharmaceuticalcompositions comprising in combination an effective amount to treat apatient with autoimmune disease of a plurality of two or more componentsselected from the group consisting of: anti-IFNα antibodies, antibodiesto IFNα receptor, anti-IFNγ antibodies, antibodies to IFNγ receptor,anti-TNF antibodies, antibodies to TNF receptor, and antibodies to anHLA class II antigen or its receptor, and a pharmaceutically acceptablecarrier therefor.

It is a further object of the invention to provide kits or compositionscomprising an immunosorbent comprising an effective amount toextracorporeally remove, reduce or neutralize one or more autoimmunogensfrom the fluid of a patient with autoimmune disease of at least one ofthe following antibodies: anti-IFNα antibodies, antibodies to IFNαreceptor, anti-IFNγ antibodies, antibodies to IFNγ receptor, anti-TNFantibodies, antibodies to TNF receptor, antibodies to an HLA class IIantigen or to its receptor, antibodies to IgE. While it is yet anotherobject to provide kits or compositions comprising an immunosorbentcomprising an effective amount to extracorporeally remove, reduce orneutralize one or more auto-antibodies from the fluid of a patient withautoimmune disease of at least one of the following: target cells, CD4cells, and DNA.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art on examination of thefollowing, or may be learned by practice of the invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides methods for treating various autoimmunediseases, including AIDS by blocking, neutralizing or inhibitingdifferent kinds of interferons, tumor necrosis factor, HLA class IIantigens, and other pathological factors, which are common in mostautoimmune disease, and which participate in damage of the immune systemand the development of autoimmune disease. In addition, it providesmethods for removing, reducing or neutralizing antibodies which maydestroy the DNA or target cells of autoimmune disease patients and/orthe CD4 cells in patients with AIDS.

Interferon is now known to be not only an antiviral andanti-proliferative cytokine, but it is also a factor which plays animportant role in normal and pathological immunity. For the normalfunctioning of the immune system, it is necessary for an individual tohave a normally functioning cytokine system. The interferon system inhumans is a very stable system. Since healthy people do not haveinterferon in their blood, prolonged hyperproduction ofinterferon—primarily alpha but sometimes gamma interferons—typicallyindicates the presence of immune disease.

Upon observation of the diverse clinical pictures manifested in patientswith various autoimmune diseases, which includes hypersensitivity of theimmediate type (e.g., bronchial asthma, which is also an autoimmunecondition), and AIDS (a viral disease with autoimmune components), itbecomes apparent that these diseases have in common a large number ofsimilar laboratory characteristics. This suggests that a similar diseasemechanism is occurring in each autoimmune disease, but in differenttarget cells. Thus, it is the unique target (e.g., skin, joints, liver,and the like) of each autoimmune disease that leads to itscharacterization in terms of clinical manifestations. For example, anautoimmune attack destroying the insulin producing beta-cells of theislets of Langerhans of an individual would be diagnosed as diabetes(Type I), whereas autoimmune destruction of the conducting fibers of thenervous system is characteristic of multiple sclerosis, or autoimmunedestruction of the joint lining tissue is characteristic of rheumatoidarthritis. Likewise in the case of skin transplantation, the skin areacan be damaged. Yet in each case, the mechanism underlying theautoimmune response is similar; a high level of IFNs, a detectable levelof TNF, an elevated level of HLA class II antigens in the blood or onthe surface of the cells, and antibodies to target cells. In addition,cells taken from autoimmune patients show a decreased production of IFNsin vitro, even after stimulation with an interferonogen. Consequently,the method of treatment of the various autoimmune diseases is similar inprinciple, despite the apparent clinical differences among the diseases.

The present invention is based upon the inventors' conclusions that theoptimal treatment of each different autoimmune disease or autoimmunecondition involves the removal, neutralization or inhibition of complexpathological agents (including hyperproduced cytokines) from thepatient, and/or the administration to the patient of an effective amountof selected molecules or antibodies, or their receptors, to bind to,neutralize or inhibit the circulating pathological agents and/or thoseon the surface of the cells targeted in the specific autoimmune response(“target cells”). The primary indicator of each autoimmune disease isthe hyperproduction of IFNα or, to be more exact, the disturbance of thesynthesis of one or more alpha IFNs (IFNα comprises at least 15 distinctsubtypes). In most patients with autoimmune disease, some level of IFNγis also found. Patients with systemic lupus erythematosus (“SLE”) andAIDS appear to have the highest levels of IFNα, as compared withpatients with other autoimmune diseases (See, Skurkovich et al., Annalsof Allergy 35:356 (1975); DeStefano et al., 1982).

IFNα is secreted by somatic cell and leukocytes, accumulating on themembranes of cells and entering the bloodstream. In autopsies, IFNα hasbeen found, for example, on the surface of cells in the pancreas ofpatients with insulin dependent diabetes (Foulis et. al, Lancet 2:1423(1987)), in skin lesions of patients with psoriasis (Livden et. al.,Arch Dermatol. Res. 281 :392 (1989)), on the surface of brain cells ofpatients with the psychiatric complications of systemic lupuserythematosus (“SLE”) ((Shiozawa et al., Arthr. Rheum. 35:417 (1992)),and in the circulating body fluids of animal and human patients withautoimmune disease ((Skurkovich et al. 1975; DeStefano et al. 1982). Forinstance, IFNα has been found circulating in the blood of autoimmuneNZB/W and mrl/lpr mice (Skurkovich et al. Ann. Internat'l Congress forInterferon Research (1981), and in the circulation of patients with RA,SLE, Sjogren's syndrome, scleroderma, insulin-dependent diabetes,bronchial asthma, AIDS, and other autoimmune diseases (Skurkovich et.al., 1975; Hooks et al., N Engl J. Med 301:5 (1979); DeStefano et al.1982). Of particular interest, is a recent discovery that interferon isalso found in the blood and spinal fluid of patients with neurologicaldiseases, including, e.g., schizophrenia (Lebikova et al., Med MicrobiolImmun. 166:355 (1978); Preble et al., 1985), depression, and multiplesclerosis (Link et al., Ann. Neurol. 36:379 (1994)).

The uninterrupted production of IFNα is apparently connected with theweakening or absence of the IFNα repressor. In general, hyperproductionof IFNα is an indicator of immunological disintegration, and manyscientists consider IFNα to be a recognized marker of the presence of anautoimmune condition ((Skurkovich et al., 1975; Hooks el ail. 1979).Moreover, the disturbance of IFNα production in an individual changesthe biological activity of the cells, bringing about the production ofautoantigens (Skurkovich et al., 1994; Shattner et al. Am. J. Med. Sci.295:532 (1988)). The hyperproduction of IFNα also stimulates theproduction of tumor necrosis factor and its receptors, particularly TNFα(Lau et al., 1991). Increased production of autoantigens leads to theactivation of the T-cells, and to the production of IFNγ. It is possibleevery autoantigen stimulates the induction of a unique, specific IFNγ.

In addition, in human autoimmune disease some cells express abnormallyelevated levels of HLA class II antigens, or in some cases HLA class Ior III antigens, which is stimulated by the disturbed production ofIFNγ, alone or in combination with TNF (Feldman et. al., 1987). Thissynthesis of HLA class II antigens (or HLA class I or III antigens)plays an important role in the pathogenesis of autoimmune disease andAIDS. The disturbance of the production of HLA class II antigen in anindividual leads to a pathological disturbance of the presentation ofantigens to the T-cells, to disrupted T/B cooperation, and to thedysregulation of the interactions among T-cells.

Every antigen is an interferonogen; “self” cannot induce IFN. Thus, theproduction of IFN signals the invasion by a foreign antigen, or in thiscase the presence of an autoantigen. The production of IFN and itsprolonged circulation in the body is an inseparable part of thedevelopment of autoimmune disease, and triggers immunological chaos. Forexample, antibodies to CD4 in patients with HIV infection (Dorsett etal. Am. J. Med. 78:621 (1985)) can cross-react with HLA class IIantigen, which in turn are induced by IFNγ, or by IFNγ in combinationwith TNF, and possibly by IFNα, which induces TNF. IFNα and IFNγ arebiologically dangerous elements in certain people. If injected into ahuman or animal with a genetic predisposition to develop an autoimmunedisease, the interferons can trigger or exacerbate the autoimmunedisease in the recipient. For example, administration of IFNα, IFNγ, oran inducer of IFNα to autoimmune NZB/W and MRL/lpr/lpr mice haveresulted in an aggravation of the autoimmune response in the animal,augmented morbidity, and increased mortality (Carpenter et al. LabInvest. 23:628 (1970); Engleman et al. Arthr. Rheum. 24:1396 (1981);Heremans et al. Infect. Immun. 21 :925(1978)). Injection of one unit ofr-IFNγ into the thyroid gland of CBA mice caused autoimmune thyroiditis(Remy et al. Immunol Today 8:73 (1987)). Administration of IFNα to humanpatients with psoriasis (a disease with an autoimmune component) wasfound to exacerbate, rather than alleviate the clinical manifestationsof the disease (Quesada et al., Lancet 2:1466 (1986)). Injection ofnatural or recombinant IFNα (“r-IFNα”), and sometimes IFNγ, to cancerpatients has reportedly triggered or exacerbated autoimmune parotitis,epididymitis, and thyroiditis, SLE, RA, Graves' disease, and otherautoimmune conditions (See, e.g., Quesada et al., Clin. Oncol. 2:4234(1986); Bevan et al. Lancet 2:561 (1985); Ronnblom, et al. J lntern. Med227:207 (1990); Conlon et al., Cancer 65:2237 (1990); Machold et al., JRheum. 17:831 (1990); Schilling et al., Cancer 68:1536 (1991); Ronnblomet al., Ann. Intern. Med 115:178 (1991)). IFNα injections in patientswith different types of viral hepatitis have induced autoimmunehepatitis (See, e.g., Ohta et al. J Gastroenterol. 88:209 (1991);Fattovich et al., Brit. J. Med. Virol. 34: 132 (1991)). In addition, ithas been reported that a patient with multiple sclerosis (“MS”) givenr-IFNα subcutaneously (Larrey et al., JAMA 261:2065 (1989)), and anothergiven r-IFNγ (Paniteh et al., Lancet 1:893 (1987)) intrathecally,manifested clinical relapses at rates significantly higher thanexpected.

On the other hand, the neutralization of individual cytokines, such asINFα or TNFα, from the blood has been associated with a significanttherapeutic effect, in patients with RA and in patients with AIDS(Skurkovich et. al. 1975; Gringeri et al., 1996). Thus, it is a purposeof the present invention to provide methods of treating autoimmunedisease by the use of pleiotrophic autoimmune inhibitors, acting on eachof the known aberrant cytokine pathways in the patient and/or removingpathogenic cytokines, HLA antigens, or autoantibodies from theautoimmune patient.

The terms “patient” and “individual” are interchangeably used to mean awarm-blooded animal, such as a mammal, suffering from a disease, such asan autoimmune disease or “graft versus host” disease, or is in danger ofrejection of a transplanted allogeneic tissue or organ. It is understoodthat humans and animals are included within the scope of the term“patient” or “individual.”

“Cytokines” are intercellular mediators secreted by the lymphocytesand/or macrophages. For example, cytokines play a role in the generationof an immune response, such as in an immune response to an infection orinfectious organism. Cytokines including, for example, interferens (IFNαand IFNγ) and TNFs induce other cytokines which participate in thedevelopment of different autoimmune conditions and diseases. In thedevelopment of anti-cytokine therapy in accordance with the presentinvention, considerable emphasis has been placed on these threecytokines, because it appears that by neutralizing these key cytokines(IFNα, IFNγ and TNF), it is possible to decrease, halt or prevent thesynthesis of the cytokines induced by them. However, is certainautoimmune conditions or diseases, including IDDM and SLE, the inductionof another cytokine (interleukins, specifically IL-6) is so great andexerts such a strong pathological influence, that it is desirable toremove IL-6 together with the other cytokines.

IL-6 is made by several cells; including T-cells, B-cells, and others(Hirano et al., Clin. Immunol 62:S60 (1992)), and induces insulinitis inIDDM. In response to IFNγ and TNF, B-cells of the pancreas produce largequantities of IL-6. It is also an important pathological factor in thepathogenesis of SLE, where is has been found to be present at a highlevel. IL-6 stimulates differentiation in B-cells and hyperactivity ofT-cells (Snick et al., Ann. Rev. Immunol 8:253 (1990)). The increase inIL-6 parallels the increase of TNFα (Majer et. al., Lupus 2:359-365(1993)).

The term “autoimmune inhibitor” is used to refer to a “compound” or“compounds,” including one or more molecules, antigens, and/orantibodies (alone or in combination), which when administered in aneffective amount to a patient, binds to, neutralizes or inhibitscirculating pathological agents and/or those on the surface of targetcells, and which when placed in extracorporeal contact with thepatient's body fluids effects the removal, neutralization or inhibitionof complex pathological agents (including hyperproduced cytokines andautoantibodies). The autoimmune inhibitor may also comprise antibodiesto a receptor of the autoantigen. A “receptor” is a protein found on thesurface of a target cell or in its cytoplasm, that has a binding sitewith high affinity to a particular signaling substance (e.g., acytokine, hormone, neurotransmitter, etc.). By competitively inhibitingthe availability of the receptor with an analog or antibody to thereceptor, the immune response to the autoimmunogen is modified orneutralized.

In accordance with the present invention, treatments involvingadministration of an autoimmune inhibitor to a patient, and treatmentsinvolving the extracorporeal exposure of the patient's fluid to anautoimmune inhibitor, may be performed alone or in combination.

Administered autoimmune inhibitor of the invention binds to, neutralizesand/or inhibits the molecule(s) associated with or causing theautoimmune response in the patient. More specifically, administration ofthe autoimmune inhibitor to a patient results in suppression ofpathological humoral and adaptive immunity in the patient. In otherwords, in accordance with the method of the present invention, treatmentof a patient with the autoimmune inhibitor causes the humoral andadaptive immune response of the patient to be inhibited or neutralizedover that which was, or would have been, present in the absence oftreatment.

A patient is in need of treatment with an autoimmune inhibitor, when thepatient is suffering from an autoimmune disease, or “graft-versus-host”disease, or when treatment is needed to prevent rejection oftransplanted allogeneic tissues or organs, or when the patient hasproduced autoantibodies.

The term “autoimmune disease” refers to those disease states andconditions wherein the immune response of the patient is directedagainst the patient's own constituents, resulting in an undesirable andoften terribly debilitating condition. As used herein, “autoimmunedisease” is intended to further include autoimmune conditions, syndromesand the like. An “autoantigen” is a patient's self-produced constituent,which is perceived to be foreign or undesirable, thus triggering anautoimmune response in the patient, which may in turn lead to a chain ofevents, including the synthesis of other autoantigens or autoantibodies.An “autoantibody” is an antibody produced by an autoimmune patient toone or more of his own constituents which are perceived to be antigenic.For example, in AIDS disease the patient eventually producesautoantibodies to CD4 cells, in SLE autoantibodies are produced to DNA,while in many other types of autoimmune diseases autoantibodies areproduced to target cells (see, Table 1 for examples of specific targetcells of autoimmune diseases).

Patients suffering from autoimmune diseases including, e.g., rheumatoidarthritis, insulin-dependent diabetes mellitus, hemolytic anemias,rheumatic fever, thyroiditis. Crohn's disease, myasthenia gravis,glomerulonephritis, autoimmune hepatitis, multiple sclerosis, systemiclupus erythematosus and others, are in need of treatment in accordancewith the present invention. Treatment of patients suffering from thesediseases by administration of autoimmune inhibitor and/or removal ofcompound(s) by extracorporeal immunosorption in accordance with thepresent invention will alleviate the clinical manifestations of thedisease and/or minimize or prevent further deterioration or worsening ofthe patient's condition. Treatment of a patient at an early stage of anautoimmune disease including, e.g., rheumatoid arthritis,insulin-dependent diabetes mellitus, multiple sclerosis, myastheniagravis, systemic lupus erythematosus, or others, will minimize oreliminate deterioration of the disease state into a more seriouscondition.

For example, insulin-dependent diabetes mellitus (IDDM) is an autoimmunedisease which is believed to result from the autoimmune responsedirected against the beta cells of the islets of Langerhans whichsecrete insulin. Treatment of a patient suffering from an early stage ofIDDM prior to the complete destruction of the beta cells of the isletsof Langerhans would be particularly useful in preventing furtherprogression of the disease, since it would prevent or inhibit furtherdestruction of the remaining insulin-secreting beta cells. It isunderstood that treatment of a patient suffering from an early stage ofother autoimmune diseases will also be particularly useful to prevent orinhibit the natural progression of the disease state to more seriousstages.

The method of the present invention is applicable to autoimmunediseases, such as those given in the following Table 1 (which isintended to be exemplary rather than inclusive), and autoimmuneconditions, such as those listed following the Table.

TABLE 1 Autoimmune Diseases Disease Tissue Affected Addison's diseaseadrenal Autoimmune diseases of the ear ear Autoimmune diseases of theeye eye Autoimmune hepatitis liver Autoimmune parotitis parotid glandsCrohn's disease intestine Diabetes (Type I) pancreas Epididymitisepididymis Glomerulonephritis kidneys Graves' disease thyroidGuillain-Barré syndrome nerve cells Hashimoto's disease thyroidHemolytic anemia red blood cells Systemic lupus erythematosus multipletissues Male infertility sperm Multiple sclerosis nerve cells MyastheniaGravis neuromuscular junction Pemphigus primarily skin Psoriasis skinRheumatic fever heart and joints Rheumatoid arthritis joint liningSarcoidosis multiple tissues and organs Scleroderma skin and connectivetissues Sjogren's syndrome exocrine glands, and other tissuesSpondyloarthropathies axial skeleton, and other tissues Thyroiditisthyroid Vasculitis blood vessels

Autoimmune conditions for which the method of the present invention isapplicable include, for example, AIDS, atopic allergy, bronchial asthma,eczema, Behdet's syndrome leprosy, schizophrenia, inherited depression,transplantation of tissues and organs, chronic fatigue syndrome,Alzheimer's disease, Parkinson's disease, myocardial infarction, stroke,autism, epilepsy, Arthus's phenomenon, anaphylaxis, and alcohol and drugaddiction. In the above-identified autoimmune conditions, the tissueaffected is the primary target, in other cases it is the secondarytarget. These conditions are partly or mostly autoimmune syndromes.Therefore, in treating them, it is possible to use the same methods, oraspects of the same methods that are herein disclosed for treatingautoimmune disease, sometimes in combination with other methods.

Preferred embodiments of the invention are directed toward the treatmentof specific autoimmune disease or condition in a patient, includingthose identified herein, and particularly including RA, SLE, MS,juvenile RA, and ankylosing spondylitis.

Patients who have received, or who are about to receive, an allogeneictissue or organ transplant, such as an allogeneic kidney, liver, heart,skin, bone marrow, are also patients who are in need of prophylactictreatment with an autoimmune inhibitor and/or removal of compound(s) byextracorporeal immunosorption in accordance with the present invention.The autoimmune inhibitor of the present invention will minimize orprevent the adaptive and humoral immune response of the donee fromrejecting the allogeneic tissue or organ of the donor. Likewise, forpatients suffering from graft-versus-host disease treatment with anautoimmune inhibitor in accordance with the method of the presentinvention will minimize or prevent the adaptive and humoral immuneresponse of the transplanted tissue or organ from rejecting theallogeneic tissue or organ of the donee.

Based on standard clinical and laboratory tests and procedures, anattending diagnostician, physician or other person skilled in the art,can readily identify those patients who are in need of treatment with anautoimmune inhibitor. Such an individual can also determine the compoundor compounds to be included in the autoimmune inhibitor for treatment inaccordance with the methods of the present invention, based upon theincreased synthesis of cytokines typifying the general onset andprogression of autoimmune disease, and on the clinical manifestations ofthe particular disease being treated.

The term “fluid” refers to blood, plasma, plasma containing leukocytes,serum, serum and leukocytes, peritoneal fluid, cerebrospinal fluid,synovial fluid, amniotic fluid, or the like, drawn from the patient inthe practice of the present invention.

An effective amount of autoimmune inhibitor is that amount which iseffective, upon single or multiple dose administration to a patient, tobind to, neutralize or inhibit the autoimmunogen(s) causing (directly orindirectly) or involved with the clinical manifestation(s) of theautoimmune disease in the patient. In addition, an effective amount ofthe autoimmune inhibitor in an immunosorbent column over which thepatient's fluid is passed, is that amount which removes, neutralizes orinhibits the autoimmunogen(s) causing (directly or indirectly) orinvolved with the clinical manifestation(s) of the autoimmune disease inthe patient. The effect of administering the autoimmune inhibitor and/orof extracorporeally passing fluid from the patient over immunosorbent(s)comprising the autoimmune inhibitor in accordance with the method of thepresent invention, can be seen as a slowing, interruption, inhibition,neutralization or prevention of the adaptive immune response associatedwith the autoimmune disease, often displayed as an alleviation ofclinical manifestations of the disease. For example, theimmunosuppressive effect of administering an effective amount ofantibody to IFNγ to a patient in need of such treatment would be theinhibition or prevention of further expression of IFNγ by the patient,which could be quantitatively determined in terms of reduced fluidactivity level of one or more of the elevated cytokines, i.e., INFγ orTNF-α. The lowering of the cytokine activity level may be measureddirectly in the treated patient, or the reduction in cytokine activitylevel may be projected from clinical studies in which dose regimensuseful in achieving such reduction are established.

An effective amount of autoimmune inhibitor can be readily determined bythe use of known techniques and by observing results obtained underanalogous circumstances. In determining the effective amount or dose, anumber of factors are considered by the attending diagnostician,including, but not limited to: the species of mammal; its size, age, andgeneral health; the specific disease involved; the degree of orinvolvement or the severity of the disease; the response of theindividual patient; as well as for purposes of administration, theparticular compound being administered; the mode of administration; thebioavailability characteristics of the preparation administered; thedose regimen selected; the use of concomitant medication; and otherrelevant circumstances.

The autoimmune inhibitor of the present invention may comprise a singlecompound or anti-cytokine, e.g., anti-IFNγ antibody administered to thepatient or used in extracorporeal immunosorption, or it may be acombination of anti-cytokines or compounds, e.g., a combination ofantibodies to IFNs, TNFs, and the like, administered to the patient orused in extracorporeal immunosorption, and/or antigens such as a targetcell, including a CD4 cell, used in extracorporeal immunosorption. Whencombined, the compounds may be used concomitantly in an admixture or assimultaneous processes, or the compounds may be used sequentially toprovide a combined effect without being in physical combination. Forexample, an AIDS patient may be treated by passing his blood, plasma orthe like extracorporeally over an immunosorbent comprising CD4 cells toremove autoimmune antibodies against his own CD4 cells, while at thesame time, or sequentially, anti-cytokines may be administered toneutralize, for instance the interferons and TNFs that have been inducedwithin his body. The sequential treatments may occur in any order, solong as the autoimmune inhibitors have the desired anti-autoimmuneeffect.

Combined treatments, comprising the use of one or more autoimmuneinhibitors in accordance with a preferred embodiment of the invention,may be mechanistically advantageous. This is because althoughcirculating immunogens can be removed extracorporeally by passing thepatient's body fluid over an immunosorbent comprising the autoimmuneinhibitor(s), the administration of suitable autoimmune inhibitor(s),such as anti-cytokine antibodies, can effectively neutralize theimmunogens, such as cytokines, both in circulation and on the cellsurface. For example, to remove autoantibodies to CD4 cells, CD4 cellsmust be placed into an immunosorbent column. The body fluid from thepatient is extracorporeally exposed to an immunosorbent comprising CD4cells or their fragments, then the treated fluid (minus the antibodiesthat would otherwise attack his own CD4 cells) is returned to thepatient. An attending diagnostician, physician or other person skilledin the art, can readily identify those patients who are in need ofadministrative treatment with an autoimmune inhibitor, or those whowould benefit from extracorporeal treatment of their body fluids, orthose who would benefit from a combination of the two.

The compound(s) comprising the autoimmune inhibitor, e.g., antibodies toIFNs, TNFs, and the like, and/or antigens such as a target cell,including CD4 cells, in accordance with the methods of the presentinvention, include cytotoxic amino acid sequence and glycosylationvariants which also are used herein. The terms likewise coverbiologically active functional equivalents, derivatives, or allelic orspecies variants of each compound, e.g., those differing by one or moreamino acids(s) in the overall sequence. Further, the terms used in thisapplication are intended to cover substitution, deletion and insertionamino acid variants of each compound, or post-translationalmodifications thereof.

Removal, neutralization and/or inhibition of alpha and gamma IFNs, TNF,and HLA class II antigen, and the like, and/or their receptors can beaccomplished by the administration to the patient of one or moreantibodies, or by including one or more antibodies in the immunosorbentover which the patient's body fluid is passed for extracorporealtreatment. As used herein, the term “antibody” is intended to includemonoclonal or polyclonal antibodies, or a combination thereof, humanizedforms of the monoclonal antibodies (comprising only human antibodyprotein), and chimeric monoclonal antibodies, as well as biologicallyactive fragments, functional equivalents, derivatives, or allelic orspecies variants thereof Treatment can include polyclonal antibodiesfrom different animal species.

The term “biologically active fragment” is intended to mean a part ofthe complete molecule which retains all or some of the catalytic orbiological activity possessed by the complete molecule, especiallyactivity that allows specific binding of the antibody to an antigenicdeterminant.

“Functional equivalents” of an antibody include any molecule capable ofspecifically binding to the same antigenic determinant as the antibody,thereby neutralizing the molecule. e.g., antibody-like molecules, suchas single chain antigen binding molecules.

“Derivative” is intended to include both functional and chemicalderivatives, including fragments, segments, variants or analogs of amolecule. A molecule is a “chemical derivative” of another, if itcontains additional chemical moieties not normally a part of themolecule. Such moieties may improve the molecule's solubility,absorption, biological half life, and the like, or they may decreasetoxicity of the molecule, eliminate or attenuate any undesirable sideeffect of the molecule, and the like. Moieties capable of mediating sucheffects are disclosed in Remington's Pharmaceutical Sciences (1980).Procedures for coupling such moieties to a molecule are well known inthe art.

A “variant” or “allelic or species variant” of a protein refers to amolecule substantially similar in structure and biological activity tothe protein. Thus, if two molecules possess a common activity and maysubstitute for each other, it is intended that they are “variants,” evenif the composition or secondary, tertiary, or quaternary structure ofone of the molecules is not identical to that found in the other, or ifthe amino acid or nucleotide sequence is not identical.

The term “IFN” is intended to refer to any known subtype of IFN. Forexample, “IFNα(” is broadly intended to include any of the known 15subtypes of IFNα, or any that may be determined in the future. The term“HLA class II antigens” is intended to mean not only HLA class IIantigens, but also where appropriate, HLA class I or III antigens.

Any animal (mouse, rabbit, human, etc.) which is known to produceantibodies can be utilized to produce antibodies with the desiredspecificity. Methods for immunization are well known in the art. Suchmethods include subcutaneous or interperitoneal injection of thepolypeptide. One skilled in the art will recognize that the amount ofpolypeptide used for immunization will vary based on the animal which isimmunized, the antigenicity of the polypeptide and the site ofinjection. Chimeric antibodies, generated by recognized methods can alsobe used, including antibodies produced by recombinant methods.

If the antibody is to be administered intramuscularly or intravenouslyinto the patient, then it may be preferable to use a substantiallypurified monoclonal antibody produced in human hybridoma. Humanizedforms of the antibodies of the present invention may be generated usingone of the procedures known in the art such as chimerization or CDRgrafting. Also monoclonal antibodies of completely human protein may beapplied. Until a satisfactory partner for human B-cells or activatedhuman B-cells suitable for fusion become more readily available, arecognized procedure based upon immortalization of human B-cells withEpstein-Barr virus has provided a source of human antibodies (see,Burton, Hospital Practice (August 1992), 67).

In general, techniques for preparing monoclonal antibodies are wellknown in the art (Campbell, A. M. “Monoclonal Antibody Technology:Laboratory Techniques in Biochemistry and Molecular Biology.” ElsevierScience Publishers, Amsterdam, The Netherlands (1984); St. Groth et al.,J. Immunol. Methods 35:1-21 (1980). For example, in one embodiment anantibody capable of binding to IFNγ is generated by immunizing an animalwith natural, synthetic or recombinant IFNγ.

To produce the antibodies of the present invention, a cytokine orantigen may be modified or administered in an adjuvant in order toincrease the peptide antigenicity. Methods of increasing theantigenicity of a polypeptide are well known in the art. Such proceduresinclude coupling the antigen with a heterologous protein (such asglobulin or P-galactosidase) or through the inclusion of an adjuvantduring immunization.

For monoclonal antibodies, spleen cells from the immunized animals areremoved, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, andallowed to become monoclonal antibody producing hybridoma cells. Ahybridoma is an immortalized cell line which is capable of secreting aspecific monoclonal antibody. Any one of a number of methods well knownin the art can be used to identify the hybridoma cell which produces anantibody with the desired characteristics. These include screening thehybridomas with an ELISA assay, western blot analysis, orradioimmunoassay (Lutz et al., Exp. Cell Res. 175: 109-124 (1988)).Hybridomas secreting the desired antibodies are cloned and the class andsubclass are determined using procedures known in the art (See.Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry,and Molecular Biology, supra).

For polyclonal antibodies, antibody containing antisera is isolated fromthe immunized animal and is screened for the presence of antibodies withthe desired specificity using one of the above-described procedures.Polyclonal antibodies raised from animals immunized with specificantigens (IFNs, TNF, etc.) can be used after the isolation of the activefraction (e.g., IgG) or isolated Fab fragment.

The autoimmune inhibitor antibody(ies) also may be produced and/orisolated from discordant animal species. For example, porcine or bovineantibodies may be used for the treatment of humans. To useanimal-derived antibodies for a prolonged period, antibodies from avariety of different animal species must be used, permitting the sourceof the antibodies to be changed if the patient develops ahypersensitivity or deleterious response to a component of theoriginally administered antibody, antibody fragment or polypeptide. Insome cases, to prevent allergenic reaction between injections ofantibodies from a discordant species, immunodepressant drugs, such assteroid hormones or cyclophosphamide are administered. A preferredcompound of the present invention is derived from a mature compound fromrecombinant microbial cell culture, prepared, isolated and substantiallypurified in accordance with known techniques. A combination ofmonoclonal and polyclonal antibodies can also be utilized.

To evaluate the antibody or antibodies, conditions for incubating theantibody or antibodies with a test sample vary. Incubating conditionsdepend on the format employed in the assay, the detection methodsemployed, the nature of the test sample, and the type and nature of theantibody used in the assay. One skilled in the art will recognize thatany one of the commonly available immunological assay formats (such as,radioimmunoassays, enzyme-linked immunosorbent assays, diffusion basedOuchterlony, or rocket immunofluorescent assays, or the like) canreadily be adapted to employ the antibodies of the present invention.

Autoimmune inhibitor(s) of the present invention include polypeptidescomprising the epitope of the antibody or biologically active fragmentthereof, or polypeptide that is functional in conferring protection inthe individual suffering from autoimmune disease, or functionallyconserved fragments or amino acid variants thereof. Identification ofthe epitope is a matter of routine experimentation. Most typically, onewould conduct systematic substitutional mutagenesis of the compoundmolecule while observing for reductions or elimination of cytoprotectiveor neutralizing activity. In any case, it will be appreciated that dueto the size of many of the antibodies, most substitutions will havelittle effect on binding activity. The great majority of variants willpossess at least some cytoprotective or neutralizing activity,particularly if the substitution is conservative. Conservative aminoacid substitutions are substitutions from the same class, defined asacidic (Asp, Glu), hydroxy-like (Cys, Ser, Thr), amides (Asn, Gin),basic (His, Lys, Arg), aliphatic-like (Met, Ile, Leu, Val, Gly, Ala,Pro), and aromatic (Phe, Tyr. Trp).

Homologous antibody or polypeptide sequences generally will be greaterthan about 30 percent homologous on an identical amino acid basis,ignoring for that purposes of determining homology any insertions ordeletions from the selected molecule in relation to its native sequence.The compounds discussed herein, i.e., autoimmune inhibitors foradministration to the patient with autoimmune disease and/or forremoval, neutralization or inhibition of the autoimmunogen(s) byextracorporeal immunosorption in accordance with the present invention,also include glycosylation variants as well as unglycosylated forms ofthe agents, fusions of the agents with heterologous polypeptides, andbiologically active fragments of the agents, again so long as thevariants possess the requisite neutralizing or cytoprotective activity.

The autoimmune inhibitor antibody(ies) is also effective whenimmobilized on a solid support. Examples of such solid supports include,but are not limited to, plastics such as polycarbonate, complexcarbohydrates such as agarose and sepharose, and acrylic resins, such aspolyacrylamide and latex beads. Techniques for coupling antibodies tosuch solid supports are well known in the art (Weir et. al., “Handbookof Experimental Immunology” 4th Ed., Blackwell Scientific Publications,Oxford, England, Chap. 10 (1986); Jacoby et al., Meth. Enzym. 34Academic Press, N.Y. (1974).

Additionally, one or more of the antibodies used in the above describedmethods can be detectably labeled prior to use. Antibodies can bedetectably labeled through the use of radioisotopes, affinity labels(such as, biotin, avidin, etc.), enzymatic labels (such as, horse radishperoxidase, alkaline phosphatase, etc.) fluorescent labels (such as,FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures foraccomplishing such labeling are well-known in the art, for example SeeStemberger et al., J Histochem. Cytochem. 18:315(1970); Bayer et al.,Meth. Enzym. 62:308 (1979); Engval et al., Immunol 109:129 (1972);Goding, J. Immunol Meth. 13:215 (1976). The labeled antibodies of thepresent invention can be used for in vitro, in vivo, and in sites assaysto identify cells or tissues which express a specific cytokine orantigenic protein.

For administration purposes, an effective amount of autoimmune inhibitoris expected to vary from about 0.1 milligram per kilogram of body weightper day (mg/kg/day) to about 500 mg/kg/day. Preferred amounts areexpected to vary from about 1 to about 50 mg/kg/day. Humanizedmonoclonal antibodies can be administered daily for one or more weeks,depending on need; whereas polyclonal antibodies can be given for nomore than 5-6 days. If antibodies are used from a variety of species, adifferent antibody can be given every 5-6 days.

Cytokines and other pathological agents can also be neutralized orremoved from the patient in accordance with the methods of the presentinvention by administering vaccines against the cytokines or agents.However, vaccines may be dangerous to use in vivo, unless the antibodiesthat may be induced by the treatment can be controlled. Otherwise, suchvaccines, although initially effective, may lead to immunologicaldisaster in the patient.

In effecting treatment of a patient, an autoimmune inhibitor can beadministered in any form or mode which makes the compound bioavailablein effective amounts, including oral and parenteral routes. For example,autoimmune inhibitors can be administered by inhalation, orally,subcutaneously, intramuscularly, intravenously, transdermally,intranasally, rectally, and the like. Parenteral administration isgenerally preferred. One skilled in the art of preparing formulationscan readily select the proper form and mode of administration dependingupon the particular characteristics of the compound selected, thedisease state to be treated, the stage of the disease, and otherrelevant circumstances.

The autoimmune inhibitor can be administered alone, or in the form of apharmaceutical composition in combination with pharmaceuticallyacceptable carriers or excipients, the proportion and nature of whichare determined by the solubility and chemical properties of the compoundselected, the chosen route of administration, and standardpharmaceutical practice. The compounds of the invention, while effectivethemselves, may be formulated and administered in the form of theirpharmaceutically acceptable acid addition salts for purposes ofstability, convenience of crystallization, increased solubility and thelike.

In one embodiment, the present invention provides a method of treatmentin which the autoimmune inhibitor is admixed or otherwise associatedwith one or more inert carriers. These compositions are useful, forexample, as assay standards, as convenient means of making bulkshipments, or as pharmaceutical compositions. An assayable amount of anautoimmune inhibitor is an amount which is readily measurable bystandard assay procedures and techniques as are well known andappreciated by those skilled in the art. Assayable amounts of theautoimmune inhibitor will generally vary from about 0.001% to about 75%,of the composition by weight. Inert carriers can be any material whichdoes not degrade or otherwise covalently react with an autoimmuneinhibitor. Examples of suitable inert carriers include water, aqueousbuffers, such as those which are generally useful in High PerformanceLiquid Chromatography (HPLC) analysis; organic solvents, such asacetonitrile, ethyl acetate, hexane and the like; and pharmaceuticallyacceptable carriers or excipients.

More particularly, in accordance with the present invention,pharmaceutical compositions are provided comprising an effective amountof autoimmune inhibitor in admixture or otherwise in association withone or more pharmaceutically acceptable carriers or excipients.

The pharmaceutical compositions are prepared in a manner well known inthe pharmaceutical art. The carrier or excipient may be a solid,semi-solid, or liquid material which can serve as a vehicle or mediumfor the active ingredient. Suitable carriers or excipients are wellknown in the art. The pharmaceutical composition may be adapted fororal, parenteral, or topical use, and may be administered to the patientin the form of tablets, powders, granules, capsules, suppositories,solution, suspensions, or the like.

The compounds of the present invention may be administered orally, forexample, with an inert diluent or with an edible carrier. They may beenclosed in gelatin capsules or compressed into tablets. For the purposeof oral therapeutic administration, the compounds may be incorporatedwith excipients and used in the form of tablets, troches, capsules,elixirs, suspensions, syrups, wafers, chewing gums and the like. Thesepreparations should contain a measurable amount of autoimmune inhibitoras the active ingredient, but the amount may vary depending upon theparticular form and may conveniently be between about 1% to about 90% ofthe weight of the pharmaceutical composition. The amount of the compoundpresent in compositions is such that a suitable dosage will be obtained.Preferred compositions and preparations according to the presentinvention are prepared so that an oral dosage unit form contains between5.0-300 milligrams of an autoimmune inhibitor of the invention. Dosage,in tablet or capsule form, is at a preferred dose of 1 to 25 mg/kgpatient body weight per day. The dose may be increased or decreasedappropriately depending on the response of the patient, and patienttolerance.

The tablets, pills, capsules, troches and the like may also contain oneor more of the following adjuvants: binders such as microcrystallinecellulose, starch paste, gum tragacanth or gelatin; excipients such asstarch or lactose, disintegrating agents such as alginic acid, cornstarch and the like; lubricants such as magnesium stearate; glidantssuch as colloidal silicon dioxide; and sweetening agents such as sucroseor saccharin may be added or a flavoring agent such as peppermint,methyl salicylate or orange flavoring, of the types usually used in themanufacture of medical preparations. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier such as polyethylene glycol or a fatty oil. Other dosageuniforms may contain other various materials which modify the physicalform of the dosage unit, for example, as coatings. Thus, tablets orpills may be coated with sugar, shellac, or other enteric coatingagents.

For use in oral liquid preparation, the compound(s) may be prepared as aliquid suspension, emulsion, or syrup, being supplied either in liquidform or a dried form suitable for hydration in water or normal saline. Asyrup may contain, in addition to the present compounds, sucrose as asweetening agent and certain preservatives, dyes and colorings andflavors.

Materials used in preparing these various compositions should bepharmaceutically pure and non-toxic in the amounts used. As used herein,a protein is said to be “pharmaceutically pure” if the autoimmuneinhibitor comprises no substance that would be harmful to the patient. A“substantially pure” or “substantially purified” protein is one in whichspecific activity cannot be significantly increased by furtherpurification, and if the specific activity is greater than that found inwhole cell extracts containing the protein.

The method of the present invention is also accomplished by injectingthe selected compound(s) in the autoimmune inhibitor, e.g.,intravenously, intramuscularly, or subcutaneously, in the form ofaqueous solutions, suspensions or oily or aqueous emulsions, such asliposome suspensions. Typically, for parenteral administration, theextract is formulated as a lipid, e.g., triglyceride, or phospholipidsuspension, with the extract components being dissolved in the lipidphase of the suspension. These preparations should contain at least 0.1%of an autoimmune inhibitor of the invention, but may be varied to bebetween 0.1 and about 50% of the weight thereof. The amount ofautoimmune inhibitor present in such compositions is such that asuitable dosage will be obtained. Preferred compositions andpreparations according to the present invention are prepared so that aparenteral dosage unit contains between 5.0 to 100 milligrams ofautoimmune inhibitor. Dosage level may be increased or decreasedappropriately, depending on the conditions of disease, the age of thepatient, etc.

The solutions or suspensions may also include one or more of thefollowing adjuvants: sterile diluents such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl paraben; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylene diaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose.

The parenteral preparation can be enclosed in ampules, disposablesyringes or multiple dose vials made of glass or plastic.

Moreover, the invention provides for the treatment of a patient withautoimmune disease by the use (administration or use in extracorporealimmunosorbent) of one or more antisense molecules, which arecharacterized by the ability to bind to the autoimmunogen, or afunctionally equivalent derivative, or allelic or species variantthereof “Antisense sequence,” or “antisense molecule” refers to peptidesderived from pseudogenes which are constructed by reversing theorientation of the gene encoding the autoimmunogen with regard to itspromoter, so that the antisense strand is transcribed. The term alsorefers to the antisense strand of RNA or of cDNA which compliments thestrand of DNA encoding the cytokine, autoimmunogen, protein or peptideof interest.

When introduced into the patient, the anti-sense molecule binds to,neutralizes or inhibits the autoimmunogen, much the same as an antibody.Thus, the present methods can be practiced by means of one or moreantisense molecules. Moreover, when the nucleic acid sequence encodingthe autoimmune anti-sense molecule is introduced into the cells underthe control of a promoter, the anti-sense gene molecule binds to,neutralizes or inhibits the gene(s) encoding the autoimmunogen(s),inhibiting or preventing further pathogenesis. The inhibition appears todepend on the formation of an RNA—RNA or cDNA-RNA duplex in the nucleusor in the cytoplasm. Thus, if the antisense gene is stably introducedinto a cultured cell, the normal processing and/or transport is affectedif a sense-antisense duplex forms in the nucleus; or if antisense RNA isintroduced into the cytoplasm of the cell, the expression or translationof the autoimmunogen is inhibited. Such antisense nucleic acid sequencesmay further include modifications which could affect the biologicalactivity of the antisense molecule, or its manner or rate of expression.Such modifications may also include, e.g., mutations, insertions,deletions, or substitutions of one or more nucleotides that do notaffect the function of the antisense molecule, but which may affectintracellular localization. Also, the nucleic acid sequence maydetermine an uninterrupted antisense RNA sequence or it may include oneor more introns.

In a particular embodiment of the invention, a unique combination ofcompounds may be combined to form the autoimmune inhibitor to be usedfor the treatment of multiple sclerosis (“MS”), for which there is noother rational treatment. The administration of beta interferon (“INFβ”)has been shown to decrease the rate of exacerbation of the disease insome patients. This positive effect can be explained by the fact thatINFβ decreases the synthesis of IFNγ and TNF (Henniger et al., Neurology46: 1633-1639 (1996)). These data both confirm the negative effect ofIFNγ and TNF on the autoimmune process, and validate the synergic actionin MS of anti-cytokine antibodies (anti-IFNγ antibodies and anti-TNFantibodies) together with the administration of the cytokine IFNβ todecrease the production of IFNγ and TNF.

In one embodiment of the invention, treatment comprises passing thefluid drawn from the patient over immunosorbent comprising theautoimmune inhibitor, followed by returning the treated fluid to itssource. This method is particularly suited for treating certainautoimmune conditions in which the autoimmune inhibitor cannot beadministered to the patient. For example, in a preferred embodiment, thepatient's fluid is exposed to an immunosorbent comprising an effectiveamount of target cells, CD4 cells, and/or DNA, to remove, neutralize orinhibit the autoantibodies in the patient's fluid, followed by returningthe treated fluid to the patient. The immunosorbent for extracorporealtreatment may further comprise one or more antibodies (e.g., anti-IFNαantibodies, antibodies to IFNα receptor, anti-IFNγ antibodies,antibodies to IFNγ receptor, anti-TNF antibodies, antibodies to TNFreceptor, antibodies to an HLA class II antigen or to its receptor, orimmunoglobulin E (“IgE”).

To counter transplant rejection, antibodies to IFNα and IFNγ, or in somecases IFNγ alone, and the antigen of the transplanted cell or organ areplaced in the immunosorbent column. To treat myocardial infarction orstroke, antibodies to IFNs and cardiac or brain antigens, respectively,are placed in the immunosorbent column. Further, the present inventionmay be used in combination with immunosuppressive therapy to achieve thedesired results.

In another preferred embodiment of the invention, the patient's fluid isextracorporeally exposed to an immunosorbent comprising target cells.For example, for the treatment of rheumatoid arthritis, target cellantigens from joints, skin, collagen, and possibly other targetantigens, are used as immunosorbents, alone or in conjunction with otherautoimmune inhibitors, such as antibodies to IFNs and/or TNF or theirreceptors. In addition, for the treatment of rheumatic fever, theinvention provides an immunosorbent comprising antibodies to IFNs and/orTNF or their receptors and/or other substances, in conjunction with asecond cardiac tissue sorbent for removing autoantibodies againstcardiac tissue. The second sorbent can also include selected serotypesof Streptococcus (group “A”), because certain antigens from cardiactissue and some serotypes of Streptococcus are antigenically similar.For the treatment of autoimmune diseases of the central nervous system,target cell antigens from brain cells, e.g., to nuclear, membrane orcytoplasm antigens, are used to absorb autoantibodies formed against thebrain cells.

In yet another preferred embodiment of the invention, the patient'sfluid is extracorporeally exposed to an immunosorbent comprising DNA.For example, for the treatment of SLE the immunosorbent comprises DNA toremove, reduce or neutralize the patient's anti-DNA autoantibodies. Fora description of anti-DNA antibodies as they appear in SLE, SeeGraninger et. al., J. Rheumatol. 8:1621-1622 (1981).

In a further preferred embodiment the fluid is extracorporeally exposedto an immunosorbent comprising antibody to IgE. For example, fortreating certain diseases related to hypersensitivity of the immediatetype, e.g., bronchial asthma, antibody to IgE is used as animmunosorbent, alone or in conjunction with other autoimmune inhibitors,such as antibodies to IFNs and/or TNF or their receptors.

In an additional preferred embodiment of the invention the patient'sfluid is extracorporeally exposed to an immunosorbent comprising CD4cells. For example, for the treatment of AIDS, the immunosorbentcomprises CD4 cells, alone or in conjunction with other autoimmuneinhibitors, such as antibodies to IFNs and/or TNF and/or HLA class IIantigen, or their receptors. The CD4 component of the immunosorbentcomprises lymphocytes, primarily CD4 cells, from healthy donors toabsorb serum autoantibodies which react with the patient's own CD4cells.

For extracorporeal treatment, the pathogenic antibodies and/or immunelymphocytes can be removed or reduced by passing any of the previouslydescribed fluids over the prepared immunosorbent column comprising anautoimmune inhibitor. When using whole blood, plasma, or plasma withleukocytes, one can use a blood cell separator (e.g., Cobe “Spectra”) towhich the immunosorbent column is connected. See, e.g., U.S. Pat. No.4,362,155, which is incorporated herein by reference. To removepathological substances from joint or spinal fluids or the like, aspecial extracorporeal device with a small amount of immunosorbent isused. To neutralize antibodies to autoimmunogens, such as antibodies totarget cells, including CD4 cells, the cells themselves or that portionof the cells containing the antigenic determinant(s) for the subjectantibodies, must be placed directly in the immunosorbent column.

For the removal of compound(s) by extracorporeal immunosorption inaccordance with the present invention, particles of sorbent material,such as amorphous silica or Sepharose, can be readily placed in acontainer to prepare the immunosorbent for the extracorporeal procedure.The container can be constructed of any material which can readilyundergo steam, chemical, or gamma-irradiation sterilization. Forinstance, glass, polycarbonate, polystyrene, polymethylmethacrylate,polyolefins such as polyethylene and polypropylene, are all suitable.

Various ways of retaining or immobilizing sorbent material within acontainer are available. For instance, sorbent material may be placedbetween layers of retaining filters, or placed within a porous solidmatrix. The solid matrix immobilizes the sorbent, while simultaneouslypermitting flow of blood or other fluids, and contact with the sorbent.As is readily apparent to one of ordinary skill in the art, a widevariety of structures are available for providing suitable fluid/sorbentcontact, structures which do not cause significant hemolysis. Prudentuse of additional filters to retain the sorbent particles in theircontainer is preferred. The pretreated, immobilized sorbent may becontacted with the fluid in a variety of ways, e.g., admixture, elution,and the like, which would be recognized in the art.

Although a columnar sorbent bed is exemplified in Example 1, beds of anyother shape capable of functioning in the manner described herein mayalso be used. The length-to-diameter ratio of the sorbent bed should beselected so as to minimize any pressure drop along the bed, and toensure that shear rates remain below the known values that correlatewith cellular damage or destruction. The pressure drop along the sorbentbed (and thus the increase in shear rate) is directly proportional tothe length of the bed. However, mitigating against use of a short bed isthe fact that clearance of a substance from the fluid increases with alonger bed. The capability of the sorbent to adsorb can be assessed byexperiments in which a test solution (such as whole blood or plasma) iscontacted with the prepared sorbent at a constant temperature. The datagenerated from such an experiment can be used to determine anequilibrium constant (K), according to which the capacity of theprepared sorbent is determined. An equilibrium constant (K) is definedin units of (ml solution/g composition). The capacity of a compositionprovides a way to estimate the mass of the prepared sorbent required toremove a certain quantity of material, such as a cytokine, fromsolution.

In one embodiment of the invention, one skilled in the art will readilyrecognize that the disclosed autoimmune inhibitor or immunosorbentcomprising the autoimmune inhibitor of the present invention can readilybe incorporated into one of the established kit formats which are wellknown in the art. While in yet another embodiment of the presentinvention, kits are provided which contain the necessary reagents tocarry out the previously described methods. For example, in one instancesuch a kit comprises a pharmaceutical composition or antibody cocktailcomprising the necessary autoimmune inhibitor, with or withoutpharmaceutically acceptable carriers, excipients and the like, in anamount suitable for administration to a patient suffering from anautoimmune disease. In another instance, such a kit comprises theautoimmune inhibitor bound to an immunosorbent that may be used for theextracorporeal treatment of autoimmune disease in a patient. Inparticular, such a kit comprises an effective amount to extracorporeallyremove, reduce or neutralize one or more autoimmunogens from the fluidof a patient with autoimmune disease of at least one of the following:anti-IFNα antibodies, antibodies to IFNα receptor, anti-IFNγ antibodies,antibodies to IFNγ receptor, anti-TNF antibodies, antibodies to TNFreceptor, antibodies to an HLA class II antigen or to its receptor,and/or antibodies to IgE. Another preferred kit comprises an effectiveamount to extracorporeally remove, reduce or neutralize one or moreautoantibodies from the fluid of a patient with autoimmune disease of atleast one of the follows ing: target cells, CD4 cells, or DNA. While,yet additional kits comprise components of each of the previouslydefined kits, to provide the combined treatments of the presentinvention.

All essential publications mentioned herein are hereby incorporated byreference.

In order that those skilled in the art can more fully understand thisinvention, the following examples are set forth. These examples areincluded solely for the purpose of illustration, and should not beconsidered as expressing limitations unless so set forth in the appendedclaims.

EXAMPLES

In the following examples and protocols, all commercially availablereagents were utilized in accordance with the manufacturer'srecommendations. The cell and protein purification methods utilized inthis application are established in the art and will not be described indetail. Methodologic details may be readily derived from the citedpublications.

Example 1 Preparation of the Immunosorbent Column

Using a column and tubing made of plastic approved for the use of blood,a column is prepared of small total volume, approximately 30-35 ml. Thecolumn is filled with immunosorbent, consisting essentially of one ormore antigens or antibodies bound to Sepharose 4B or another suitablematrix, through a short filling tube placed at one end of the column.After the column has been filled, an input tube to introduce the fluidsample, and a return tube to return the treated sample to its source,are connected to either end of the column. A filter is interposedbetween the input tube and the column, and a second filter is interposedbetween the column and the return tube. The two filters prevent the flowof immunosorbent from the column. Two way stopcocks on the tubesregulate flow throughout the system.

Sepharose CL-4B (Pharmacia, Piscattaway, N.J.) (100 ml) is washedthoroughly with pyrogen free water, then suspended in 300 ml ice cold 1M NaCO₃ pH 11.0. 20 gms CNBr in 10 ml acetonitrile is added to theSepharose. After 2 minutes this is collected on a fretted glass funnel.The Sepharose cake is washed with 5 volumes of ice cold 0.2 M NaBicarbonate buffer, pH 9.5, and 5 volumes of ice cold 0.5 M NaBicarbonate buffer, pH 8.5.

The prepared Sepharose is immediately resuspended in a solution of theselected antigen or antibody or combination of one or more antigensand/or antibodies. In this case, the immunosorbent column isspecifically prepared to bind to IFNα, so the prepared Sepharose isresuspended in a solution of 780 mg anti-alpha IFN antibody in 200 ml of0.2 M Bicarbonate buffer, pH 9.3. This is incubated for 20 hours at 4°C. This is then centrifuged, the supernatant is decanted, and sedimentis resuspended in 100 ml of 0.05 PBS (phosphate buffered saline) and 2 Mglycine, pH 8.0, for 12 hours at room temperature. This is then washedthoroughly with 20 volumes of PBS.

The column is positioned lower than the source of the fluid sample,whereupon the fluid drawn from the patient flows into the column underthe influence of gravity. After the fluid perfuses through theimmunosorbent, it is collected in a holding tube from which it isreturned to the source of the fluid.

Example 2 Production of Antibody to Human IFNγ

Adult rabbits are immunized with purified human IFNγ (10⁵-10⁶ unit/mgprotein). The interferon is first mixed with equal volumes of Freund'sComplete Adjuvant and 30% Arlacel A and injected IM or subcutaneously onday 1, 4, 14 and 43 (100 units, 200 units, 200, 200 respectively). Next,200,000 units of the interferon is injected per month, for an additional6 months. The serum is drawn from the rabbit when the titer has reached100 units (1 unit of antibody neutralizes 10 units of gamma IFN), afterwhich IgG is isolated and substantially purified in accordance withrecognized methods.

Example 3 Responses to TNFα, IFNα, and IFNγ Antibodies, Separately andTogether, in Patients with Active Rheumatoid Arthritis

Polyclonal antibodies were obtained by immunizing sheep with naturalhuman IFNα, and goats with recombinant human IFNγ (“r-Hu-IFNγ”) orrecombinant human TNFα (“r-Hu-TNFα”), and isolating the IgG from theanimals. Each milliliter of IgG contained approximately 50 mg ofprotein, and the antibodies showed a 1:5 signal to noise ratio at 1:1250(anti-IFNα antibodies) and 1:12,500 (anti-IFNγ antibodies and anti-TNFαantibodies) dilutions by ELISA (Cytolmmune Sciences, Inc.). Afterobtaining approval and informed consent, 20 human patients with verysevere rheumatoid arthritis (“RA”), aged 27-64, average disease duration9 years, were equally randomized to one of four (4) treatment groups.The patients in Group A, B and C were given one intramuscularadministration of 2-3 ml/day for 5 consecutive days of (Group A)anti-TNFα antibodies; (Group B) anti-IFNα antibodies; or (Group C)anti-IFNγ antibodies. The patients in Group D were given a combinationof anti-TNFα antibodies+anti-IFNα antibodies +anti-IFNγ antibodies (6ml/day—2 ml of each antibody). All patients met the criteria of theAmerican College of Rheumatology for the diagnosis of RA and had notresponded to any of the standard disease-modifying rheumatoid drugs.Other criteria for entry into the study included radiographic evidenceof bone erosion, the presence of severe illness as indicated by thepresence of 6 or more swollen joints and 3 of 4 secondary indicationsincluding 45 minutes or more of continuous morning stiffness, 6 or morepainful joints, erythrocyte sedimentation rate (ESR) of 25 mm/hr orhigher, and C-reactive protein of 20 mg/l or higher. Patients who werepregnant or who had serious illnesses or conditions such as anemia,leukopenia, marked ankylosis of the joints were excluded.

The primary response was determined by the Paulus index (Paulus et al.,Arthritis Rheum. 33:477-484 (1990)), i.e., ≧20% or ≧50% improvement in≧4of 6 measures of laboratory and clinical effects (Table 2), which wereobtained through day 28. These include morning stiffness, number ofpainful and inflamed joints, ESR, and at least a 2-point improvement ona 5-point scale of disease severity assessed by patient and byphysician. To maintain consistency, the same physician was used to makeall assessments.

Results

Signs of inflammation dropped in some patients within each group on dayone. All groups demonstrated marked improvement by day 7, thoughindividual variation appeared in each treatment group. Table 2 shows theproportion of patients achieving≧20% improvement in the Paulus measures.Based on these 6 measures, the most positive response for all treatmentgroups was in the number of swollen and painful joints. At day 7, thepositive responses using anti-TNFα antibodies (Group A), and thecombined antibody treatment (antibodies to all three cytokines; GroupD), were the strongest. Three (3) of the five (5) patients receivinganti-TNFα antibodies, and two (2) of the five (5) receiving the combinedantibody treatment achieved≧20% improvement in 4 or more Paulusmeasures, and at least one patient in each group achieved at least 50%improvement.

In both Group A and D, all patients had at least 20% improvement inmorning stiffness and reduction in the number of painful and swollenjoints. Three (3) of the five (5) patients in both groups reported atleast a 2-point reduction (on a 5-point scale) in overall diseaseseverity. At day 28, the response to anti-IFNγ antibodies (Group C) wasthe strongest, including one (I) patient reporting at least 50%improvement, and two (2) others reporting at least 20% improvement in atleast 4 of the 6 measures. In Group D (the combined antibody therapy),two (2) patients reported at least 20% improvement in 4 or moremeasures. By comparison, at day 28 only 1 of 4 patients in Group A (theanti-TNFα antibody treatment group) reported having at least 20%improvement in 4 of the 6 measures. Comparable results are achieved byextracorporeal immunosorption as defined above, or by extracorporealimmunosorption in conjunction with administration of an autoimmuneinhibitor.

Four (4) of the 20 patients were taken off therapy or follow-up after atemporary redness appeared at the point of injection. Two (2) patientsreceiving anti-IFNα antibodies (Group B) and one patient each receivinganti-TNFα antibodies (Group A), and the combination therapy (Group D)exhibited such reactions.

TABLE 2 Proportion of Patients Achieving ≧20% Improvement in SixMeasures at Day 7 and Day 28, and Paulus Index by Treatment Group Anti-TNFα Anti-IFNγ Anti-IFNα Ab Combined Paulus Measures d.7 d.28 d.7 d.28d.7 d.28 d.7 d.28 Morning stiffness(min.) 2/S 4/5 3/4 3/3 5/5 3/4 5/53/4 No Swollen Joints 4/5 3/5 2/4 2/3 5/5 3/4 5/5 3/4 No Painful Joints4/5 4/5 2/4 3/3 5/5 4/4 5/5 3/4 Disease Severity 1/5 1/5 0/4 0/3 3/5 1/42/5 2/4 (by Physician*) Disease Severity 1/5 2/5 0/4 0/3 3/5 2/4 3/5 1/4(by_Patient*) ESR 2/5 3/5 1/4 2/3 1/5 1/4 1/5 1/4 Paufus Index ≧20%**1/5 2/5 0/4 2/3 3/5 1/4 2/5 2/4 ≧20%** 0/5 1/5 0/4 0/3 1/5 0/4 1/5 0/4*≧2-point improvement on 5-point scale as assessed by physician orpatient. **Proportion of patients achieving ≧20% (or ≧50%) improvementin ≧4 of the 6 measures at day 7 and day 28. ≧20% includes any patientachieving ≧50% improvement.

One ankylosing spondylitis (“AS”) patient, age 22, disease duration oneyear, was treated with the combined antibody regimen (antibodies toIFNα, IFNγ, and TNFα). Improvement in painful sacroiliac joint disease,diminution of radiating pain, and normalization of the erythrocytesedimentation rate was seen on days 7-8.

For repeated treatment of human patients with autoimmune disease, or fortreatment of a human patient with a secondary autoimmune condition,fully humanized monoclonal antibodies must be used or, as a temporaryalternative, chimeric monoclonal or multi-specied IgG polyclonalantibodies or active antibody fragment preparations.

The results indicate that a common mechanism appears to underlie allautoimmune diseases, with disturbed cytokine production in differenttarget cells producing the various clinical manifestations. Moreover,the results establish that each cytokine (e.g., IFNα, IFNγ, TNFα) playsits own pathological role in the mutual induction and activation ofother cytokines, suggesting a single target in treatment.

Although other autoimmune diseases may require treatment with differentanti-cytokines, antibodies or combination of autoimmune inhibitors,neutralization of such agents, e.g., the exemplified cytokines, appearsto break the chain of pathological reactions typifying autoimmunedisease and normalize the synthesis of other induced cytokines inautoimmune disease patients, including AIDS patients.

Example 4 Long-Term Improvement in Child with Juvenile RheumatoidArthritis in Response to Treatment with IFNα and TNFα Antibodies

The patient was a seven-year old girl who had been diagnosed three yearsearlier (January 1993) as having juvenile rheumatoid arthritis. (“JRA”),polyarticular form, sero-negative, after presenting with fever,arthralgias, extreme limitation of motion in the right hip joint,neutrophilia, high ESR, and anemia. The patient improved slightly on aninitial regimen of non-steroidal anti-inflammatory drugs (NSAID). Butwithin six (6) months (Fall, 1993) exacerbation of her diseasenecessitated enhancing the treatment with azathioprine, NSAIDs, and withpulse therapy using Solumedrol. The patient was maintained on weeklymethotrexate from February 1994 until July 1995, when her diseaserelapsed. However, despite increased NSAID therapy, her conditioncontinued to deteriorate. In light of the ineffectiveness ofconventional therapy, and because the disease had progressed to includehip joint involvement, which invariably leads to crippling of a child,this child became a candidate for the combined antibody treatment of thepresent invention.

As described above, and using immunological techniques, antibodies toIFNγ (“anti-IFNγ antibodies”) and antibodies to TNFα (“anti-TNFαantibodies”) were obtained by immunizing goats with r-IFNγ and r-INFα,respectively, and isolating IgG from the immunized animals. Eachmilliliter of IgG contained approximately 50 mg of protein, and theantibodies showed a 1:5 signal to noise ratio at 1:12,500 dilutions byELISA (assays performed by Cytolmmune Sciences, Inc., College Park,Md.).

Two (2) ml/day each of anti-IFNγ antibodies (3 days) and anti-TNFαantibodies (5 days) were administered parenterally to the child. By thesecond week of observation, absence of morning stiffness, elimination ofhip joint pain, and considerable increases in the level of physicalactivity, range of motion in the affected joints, and grip strength werenoted (See, Table 3). X-rays of the child showed improvement in theappearance of the femurs and hip joints, and greater delineation ofparticular spaces. Repeated testing of the child indicated a significantdrop in disease activity, as shown by clinical and laboratoryparameters, including pain, stiffness, grip strength, C-reactiveprotein, and others (See, Table 3). The improvement in clinical statusand the nearly normal range of motion in the child's hip jointspersisted into the fourth month, as shown by x-rays at regularcheck-ups. After six months (the most recent data available), damage tothe child's femurs and acetabulae were less marked as shown on x-rays,and she continued to improve in other parameters, to the point that onthe advice of an orthopedist, her joints were allowed to bear greaterweight.

TABLE 3 Dynamics of clinical and laboratory parameters in patient withJRA, After treatment with Anti-IFNγ antibodies and anti-TNFα antibodiesBefore Parameter Treatment Week 1 Week 2 Week 3 Week 4 Arthralgia score*4 2 2 0 0 Joint Stiffness (min.) 30 10 0 0 0 Grip Strength 20 44 72 6870 (mm/Hg) Angle of abduction- 15 15 20 n/a 30 rt. hip (degrees)Circumference of right 12.9 12.7 12.2 11.9 12.0 wrist (cm) ESR 6 3 8 6 6C-reactive protein (g/l) 0.6 neg neg neg neg *Scale of 0-5 where 5 ismost intense pain n/a = Not available.

These data point to a role of cytokines in autoimmune disease, and againreinforce the conclusion that a common pathological mechanism underliesclinically disparate forms of autoimmune disease. It is the differencesin the target cells affected that result in the varying clinicalmanifestations of the autoimmune response in a patient.

As demonstrated by the results produced in this child, neutralization ofcertain cytokines with antibodies can break the chain of pathologicalreactions and normalize the synthesis of other induced cytokines in thepatient. Other types of autoimmune disease can be treated by the use ofanti-cytokines, singly or in combinations, to counteract autoimmuneaggression and inflammation. Good results have been reported fromdouble-blind placebo controlled trials using chimeric monoclonalanti-TNF antibodies to treat RA (Elliott et al., Lancet, 344: 1105-1110(1994)). But until the present invention, there has been no suggestionof treatment of autoimmune diseases with anti-IFNγ antibodies, nor witha combination of anti-cytokine antibodies. Nor have the effects of suchtreatments been evaluated in clinical trials.

Given the striking long-term results produced by the present method, thecombined anti-cytokines, e.g., anti-TNFα antibodies in conjunction withanti-IFNγ antibodies, may even act synergistically.

Example 5 Treatment of Patients with Systemic Lupus Erythematosus

Human patients with systemic lupus erythematosus (SLE) were selectedafter obtaining approval and informed consent, in much the same manneras set forth in Example 3, and divided into two groups consisting of atleast four (4) patients each. The basis for selection was the patient'sfailure to respond to conventional therapy for SLE. Using polyclonalanti-IFNγ antibodies and anti-TNF antibodies in accordance with Example3, one group of patients was treated with anti-IFNγ antibodies, whilethe other group was treated with anti-IFNγ antibodies and anti-TNFantibodies. The antibodies were administered in accordance with theschedule and amounts set forth in Example 3 for 5 consecutive days.

Preliminary results, based upon at least one patient in each group,indicate that pain and swelling in joints have decreased and skinlesions have disappeared, further indicating that a common mechanismunderlies all autoimmune diseases, with disturbed cytokine production indifferent target cells producing the various clinical manifestations.

Comparable results are achieved by extracorporeal immunosorption asdefined above, or by extracorporeal immunosorption in conjunction withadministration of an autoimmune inhibitor.

Example 6 Treatment of Patients with Multiple Sclerosis

Human patients with multiple sclerosis (MS) were selected afterobtaining approval and informed consent, in much the same manner as setforth in Example 3, and divided into three groups consisting of at leastfive (5) patients each. The basis for selection was the presence ofactive MS and the patient's failure to respond to conventional therapyfor MS. Using polyclonal anti-IFNγ antibodies and anti-TNF antibodies inaccordance with Example 3, one group of patients was treated withanti-IFNγ antibodies, one group with anti-TNF antibodies, and one groupwith anti-IFNγ antibodies and anti-TNF antibodies. The antibodies wereadministered in accordance with the schedule and amounts set forth inExample 3 for 5 consecutive days, and the patients were followed for atleast two and one half (2½) months.

Results of the treatment were evaluated in terms of measuredneurological deficiencies and general patient function at the end of the2½-month period, as compared with pretreatment determinations of thesame criteria. Determinations were based upon the Disability StatusScale (DSS) devised by J. F. Kurztke, and the Functional System Scale(FSS), respectively. Decreasing numbers indicate improvement on the DSSscale, while increasing numbers indicate improvement on the FSS scale.Preliminary results indicate that improvement was most evident in thegroup treated with anti-IFNγ antibodies and in the group treated withanti-IFNγ antibodies and anti-TNF antibodies, as determined by the twoscales.

Additional studies indicate that the treatment may be further enhancedby the administration of beta interferon (IFNβ). When eight millioninternational units (IU) of IFNβ were given subcutaneously to patientsevery other day for two years, there was a decrease in the rate ofexacerbated symptoms in some patients. Consequently, an optimaltreatment of an MS patient appears to be the use of anti-IFNγ antibodiesor a combination of anti-IFNγ antibodies and anti-TNF antibodies (byadministration or by extracorporeal immunosorption, or both, as definedabove), plus the administration of an effective amount of IFNβ.

Example 7 Treatment of AIDS Patients

A pilot study has been conducted with AIDS patients which indicated thecorrelation between a reduction in serum IFN levels and improvedclinical status. In one study, four (4) patients with very high serumlevels of IFN and low levels of CD4 cells (25/mm³), when injected withanti-IFNα antibodies capable of neutralizing the circulating IFNα,reported an increased sense of well-being, energy, and appetite, and adisappearance of skin rashes as the circulating IFNα was neutralized andremoved. By corollary, when the symptoms returned in one patient 5months later, it was determined that circulating IFNα was again presentin his blood. However, following a second cycle of treatment withanti-IFNα antibodies, his condition improved as the levels ofcirculating IFNα diminished. See, Skurkovich et al., Med. Hypoth.42:27-35 (1994), herein incorporated by reference.

In light of the previously demonstrated effects of reducing circulatingIFNα in AIDS patients, and the consistently positive effect that hasresulted from the combined neutralization of IFNα, IFNγ and/or TNF inpatients with other autoimmune diseases, similar effects are seen inAIDS patients when treated with the combined antibodies of the presentinvention. However, greater reduction in the clinical manifestations ofAIDS disease in patients results from a combined therapy, including theneutralization or removal of IFNα, IFNγ and/or TNF (by administration ofantibodies to IFNα, IFNγ and/or TNF, and/or their receptors, and/or bythe extracorporeal exposure of the patient's fluid to an immunosorbentcomprising antibodies to IFNα, IFNγ and/or TNF, and/or their receptors),in conjunction with inhibition, removal or neutralization of autoimmuneautoantibodies in the patient. This is accomplished by extracorporeallyexposing the patient's fluid to an immunosorbent comprising CD4 cellsand/or target cells in an amount sufficient to remove, neutralize orinhibit autoantibodies to CD4 cells and/or to target cells in thepatient's fluid, followed by returning the fluid to the patient, inaccordance with the methods disclosed herein.

Based on the assumption that a common mechanism underlies all autoimmunedisease, and that it is the effect of the modified cytokine production,as well as the production of subsequent components of the autoimmunecascade, on different target cells that results in the various clinicalmanifestations of each specific disease or condition, the quality oflife can be improved, or even extended, in general in patients with anautoimmune disease or condition. Consequently, although the presentinvention has been described with reference to the presently preferredembodiments and examples, the skilled artisan will appreciate thatvarious modifications, substitutions, omissions and changes may be madewithout departing from the spirit of the invention.

1. A method of treating Acquired Immunodeficiency Disease in a patientcomprising administering to said patient an effective amount of acombination of an antibody to gamma interferon, an antibody to alphainterferon, and an antibody to tumor necrosis factor-alpha.
 2. Themethod of claim 1, wherein said antibody is selected from the groupconsisting of a monoclonal antibody, a polyclonal antibody, combinationsthereof, and a biologically active fragment, wherein the biologicallyactive fragment is a fragment of an antibody and binds gamma interferon,alpha interferon or tumor necrosis factor alpha.